Real-Time High-Sensitivity Reaction Monitoring of Important Nitrogen-Cycle Synthons by 15N Hyperpolarized Nuclear Magnetic Resonance

Here, we show how signal amplification by reversible exchange hyperpolarization of a range of 15N-containing synthons can be used to enable studies of their reactivity by 15N nuclear magnetic resonance (NO2– (28% polarization), ND3 (3%), PhCH2NH2 (5%), NaN3 (3%), and NO3– (0.1%)). A range of iridium-based spin-polarization transfer catalysts are used, which for NO2– work optimally as an amino-derived carbene-containing complex with a DMAP-d2 coligand. We harness long 15N spin-order lifetimes to probe in situ reactivity out to 3 × T1. In the case of NO2– (T1 17.7 s at 9.4 T), we monitor PhNH2 diazotization in acidic solution. The resulting diazonium salt (15N-T1 38 s) forms within 30 s, and its subsequent reaction with NaN3 leads to the detection of hyperpolarized PhN3 (T1 192 s) in a second step via the formation of an identified cyclic pentazole intermediate. The role of PhN3 and NaN3 in copper-free click chemistry is exemplified for hyperpolarized triazole (T1 < 10 s) formation when they react with a strained alkyne. We also demonstrate simple routes to hyperpolarized N2 in addition to showing how utilization of 15N-polarized PhCH2NH2 enables the probing of amidation, sulfonamidation, and imine formation. Hyperpolarized ND3 is used to probe imine and ND4+ (T1 33.6 s) formation. Furthermore, for NO2–, we also demonstrate how the 15N-magnetic resonance imaging monitoring of biphasic catalysis confirms the successful preparation of an aqueous bolus of hyperpolarized 15NO2– in seconds with 8% polarization. Hence, we create a versatile tool to probe organic transformations that has significant relevance for the synthesis of future hyperpolarized pharmaceuticals.


SABRE Polarization transfer method
The polarization transfer experiments that are reported were conducted in 5 mm NMR tubes that were equipped with a J. Young's tap. Samples for these polarization transfer experiments were based on a 5 mM solution of [IrCl(COD)(NHC)], co-ligand and the indicated additional substrate at the specified loading in methanol-d 4 or dichloromethane-d 2 (0.6 mL). The samples were degassed by two freeze-pump-thaw cycles prior to the introduction of parahydrogen at a pressure of 3 bar. Para-hydrogen (p-H 2 ) was produced by passing hydrogen gas over a spinexchange catalyst (Fe 2 O 3 ) at 28 K and used for all hyperpolarization experiments. This method produces constant p-H 2 with ca. 98% purity. Once filled with p-H 2 , samples were shaken vigorously in the specified polarization transfer field before being rapidly transported into the magnet for subsequent interrogation by NMR spectroscopy.

Biphasic SABRE Polarization transfer method
Samples for polarization using biphasic 1 conditions were prepared as follows.
[IrCl(COD)(NHC)] (5 mM), co-ligand, 15-crown-5 and substrate were dissolved in dichlormethane-d 2 (0.3 mL) in a 5 mm NMR tube that was equipped with a J. Young's tap. After degassing the sample using a freeze-pump-thaw method, the sample was exposed to 3 bar H 2 for 1 h prior to the subsequent addition of 0.3 mL of degassed D 2 O inside a glove box filled with N 2 . Once filled with p-H 2 , samples were shaken vigorously in the specified polarization transfer field before being rapidly transported into the magnet for subsequent interrogation by NMR spectroscopy.

Calculation of Enhancement Factors
1 H signal enhancements were calculated according to equation 1 where, E = enhancement level, SI(pol) = signal of polarized sample, SI(unpol) = signal of unpolarized (reference) sample.
Experimentally, both spectra were recorded on the same sample using identical acquisition parameters, including the receiver gain. The raw integrals of the relevant resonances in the polarized and unpolarized spectra were then used to determine the enhancement levels. The quoted values reflect the signal strength gain (fold) per proton nucleus in the specified group. The reference sample was allowed to equilibrate within the NMR spectrometer for 1-2 minutes prior to acquisition.
Heteronuclear enhancement factors were determined by comparison to either spectra obtained of high concentration solutions or spectra obtained under signal averaging. Calculations were made using standard literature methods. 2      Figure S7: Effect of polarization transfer field on the SABRE derived polarization resulting for Na 15 NO 2 in the presence of the co-ligand pyridine.

Effect of DMAP concentration on Na 15 NO signal enhancement
The effect of DMAP concentration on the 15 N NMR signal enhancement level of Na 15 NO 2 was investigated. This involved using a series of 5 mm NMR tubes containing incrementally increasing equivalents of DMAP with fixed concentrations of [IrCl(COD)(IMes)] (5 mM) and Na 15 NO 2 (125 mM) in methanol-d 4 (0.6 mL). Each sample was shaken in a −3.5 mG polarization transfer field for 20 seconds under 3 bar p-H 2 before being rapidly transferred into an NMR spectrometer for interrogation at 9.4 T. Figure S8 shows that at low concentrations of DMAP, the signal enhancements reduce. This is effect is likely to be caused by a reduction in the concentration of DMAP meaning that exchange with the active species, [Ir(H) 2 ( 15 NO 2 )(IMes)(DMAP) 2 ] slows. Increasing the number of equivalents of DMAP to 6 increased the signal enhancement. Very high loadings of DMAP act to reduce the observed signal gains, presumably, as they influence negatively the H 2 ligand exchange rate.

Effect of Na 15 NO 2 concentration of 15 N NMR signal enhancement
The effect of Na 15 NO 2 concentration on the 15 N NMR signal enhancement of Na 15 NO 2 was also investigated. A series of 5 mm NMR tubes containing incrementally increasing equivalents of Na 15 NO 2 were prepared with a fixed concentration of [IrCl(COD)(IMes)] (5 mM) and DMAP (60 mM) in methanol-d 4 (0.6 mL). Each sample was shaken in a −3.5 mG polarization transfer field for 20 seconds under 3 bar p-H 2 before being rapidly transferred into an NMR spectrometer for interrogation at 9.4 T. Figure S9 shows that the highest signal gains are achieved for lower concentration of Na 15 NO 2 and that increasing the number of equivalents of it relative to iridium reduces the signal enhancement.

Measurement of ligand loss rates using EXSY
The effect of the identity of the NHC ligand plays on the rate loss of the equatorially bound DMAP ligand from the active catalysts of type [Ir(H) 2 (NHC)(DMAP) 2 ( 15 NO 2 )] in methanol-d 4 was determined through the use of well-established EXSY methods. Integrals for the interchanging peaks in the associated 1 H EXSY spectra were obtained and converted into a percentage of the total detected signal.    Table S3: Effect of co-ligand on the 15 N NMR signal enhancement seen for 15 ND 3 when using [IrCl(COD)(IMes)] (5 mM), 15 NH 4 OH (10 eq.) and the specified co-ligand (5 eq.) in methanol-d 4 after polarization transfer at −3.5 mG field.

Reduction of Nitrate to Nitrite
An NMR tube containing a solution of [IrCl(COD)(IMes)] (5 mM), Na 15 NO 3 (25 eq), pyridine (3 eq.) in methanol-d 4 was exposed to 3 bar H 2 and placed inside an NMR spectrometer at 298 K. Over the course of 24 h, the sample was periodically interrogated to produce a series of 1 H NMR spectra. The integral value of the hydride resonance for [Ir(H) 2 ( 15 NO 2 )(IMes)(pyridine) 2 ] at δ H −21.49 was monitored ( Figure S10). These data show that reduction of nitrate to nitrite takes place under the reaction conditions. After 24 h, replenishing the H 2 atmosphere causes the reduction to continue. , pyridine (6 eq.), Na 15 NO 3 (25 eq.) in methanol-d 4 was exposed to 3 bar H 2 .

Study of the creation of phenyl diazonium without 15 N labelling.
A sample was prepared that contained 5.6 mg of NaNO 2 in methanol-d 4 and hyperpolarized by SABRE as detailed in Figure S12 (a). The resulting 15 N signal was detected with a S/N ratio of 480. This sample was then exposed to unlabelled aniline in aqueous HCl and a single scan 15 N spectrum recorded ~15 seconds later. A signal for phenyl diazonium chloride was detected with S/N 24 as shown in Figure S12 (b). We conclude that when optimized, these methods could be employed for the analysis of unlabelled materials.    5aR,6R,6aS)-1,4,5,5a,6,6a,7,8 1-15 N-Sodium azide (3.6 mg, 0.055 mmol, 1.1 eq.) was added to a solution of (1R,8S,9s)bicyclo[6.1.0]non-4-yn-9-ylmethanol (7.5 mg, 0.05 mmol, 1 eq.) in methanol-d 4 inside an NMR tube fitted with a J. Young's Tap. The NMR tube was placed in warm water for 30 min and then analysed by 1 H NMR spectroscopy. This showed completed conversion to the product had occurred. 1